Methods and devices for analyte detection

ABSTRACT

Methods for detecting one or more analytes, such as a protein, in a fluid path are provided. The methods include resolving, immobilizing and detecting one or more analytes in a fluid path, such as a capillary. Also included are devices and kits for performing such assays.

1. CROSS REFERENCES TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) to applicationSer. No. 60/589,139, entitled “Continuous Determination of CellularContents by Chemiluminescence,” filed Jul. 19, 2004 and application Ser.No. 60/617,362, entitled “Determination of Captured Cellular Contents,”filed Oct. 8, 2004, the disclosures of which are incorporated herein byreference in their entireties.

2. FIELD OF INVENTION

The present invention relates to methods, devices and kits for analytedetection and various uses thereof.

3. INTRODUCTION

Methods and devices for detecting analytes are important tools forcharacterizing analytes in biological and industrial applications. Inmany applications, it is desirable to detect the presence of one or moreanalytes in a sample. For example, rapid detection of a particularprotein in a mixture of proteins is particularly useful in molecularbiology protocols, drug development and disease diagnosis.

Although numerous approaches have been developed for detecting analytes,there is still a great need to find new assay designs that can be usedto inexpensively and conveniently detect and characterize a wide varietyof analytes. However, currently available assay protocols areinconvenient, expensive, or have other deficiencies. For example,Western blotting has been in widespread use for more than two decadesfor detecting proteins. In this technique, a sheet of gel is retainedbetween two plates and usually is mounted vertically with the upper edgeof the gel sheet accessible to the sample to be assayed. The sample isapplied in wells created along the upper edge of the gel and anelectrophoretic potential is applied between the upper and lower edgesof the sheet of gel. The electrophoretic potential is applied by a DCpower supply, and may be in the range of 50 to more than 1000 volts. Theelectrophoretic potential is applied for a period of time that allowsthe proteins in the sample, to distribute themselves (i.e., separate)vertically through the sheet of gel, typically for 1-4 hours, but insome cases considerably longer. The potential must be removed when theproteins are distributed as desired. The sheet of gel is then removedfrom between its two glass retaining plates and is then placed on asheet of blotting material such as porous nitrocellulose of length andwidth dimensions approximately matching those of the sheet of gel, theblotting material having already been soaked in a buffer to hydrate it.Care must be taken at this step to avoid the presence of air bubblesbetween the gel and the blotting material, which would impede the directtransfer of the distributed proteins from the gel to the blottingmaterial. Two electrode plates are then placed on either side of the geland blotting material, thereby sandwiching the sheets of gel andblotting material between the electrode plates. The electrode platesshould preferably apply a uniform electrophoretic field across thethicknesses of the sheets of gel and blotting material. Thiselectrophoretic field, typically 100-500 volts, transfers the proteinsfrom the gel to the blotting material in the same distribution in whichthey were captured in the gel matrix. This transfer process takesapproximately 1-2 hours, but can take as much as overnight for someproteins to be transferred. After the proteins adhere to the blottingmaterial, the blotting material is removed from the sandwich and iswashed in a buffer containing one or more blocking agents such as skimmilk, bovine serum albumin or tween-20 detergent for 1-4 hours and thenis immersed in a solution of protein-specific reporter antibodies.During the immersion the blotting paper is typically agitated by arocking or circular motion in the plane of the blotting paper. Theimmersion step typically takes 1-4 hours, but can take overnight orlonger for some antibody-protein pairs. Reporter antibody detection canbe done with a variety of markers such as optical dyes, radioactive orchromogenic markers, fluorescent dyes or reporter enzymes depending uponthe analytical method used. These results are known as Western blots asdescribed by Towbin H., Staehelin T., and Gordon J., Proc. Nat. Acad.Sci. USA, 76: 4350-4354 (1979), Bumette W. N., Anal. Biochem., 112:195-203 (1981), and Rybicki & von Wechmar in J. Virol. Methods, vol. 5:267-278 (1982).

The Western blot technique, while widely used, has a number of drawbacksand deficiencies. First, as the above description makes clear, theprocessing is very complex. There are many distinctly different steps,including the step of initially distributing the proteins being analyzedthrough the gel, the intermediate step of transferring the distributedproteins to the blotting material, the later step of binding thereporter antibodies, and the final step of reading or analyzing theresults. Between these major steps are preparation steps and washing thevariously processed components of the technique. Second, there isextensive handling of the components of the technique. The gel must beplaced in the distribution apparatus, then removed and located in theblotting apparatus, then the blotting material must be handled to bindthe reporter substrates. The components can be damaged during thishandling, in particular the fragile sheet of gel. Third, it takes aconsiderable amount of time to arrive at just a single blot. At least aday is required to produce just one blot, and generally 1½-2 days arerequired. During the beginning of the process the accuracy of thetechnique is affected by migration of the proteins until they areimmobilized in the blotting material, which can result in bandbroadening. Fourth, the variability introduced by the complexity of thehandling and processing can require the process to be repeated severaltimes before acceptable results are obtained. Fifth, the variability inthe results often requires subjective decisions to be made in readingthe results of the blot. This subjectivity reduces the ability to obtainquantifiable, objective results and frequently limits the technique topractice by highly trained and experienced personnel. Sixth, thevariability and complexity of the process impedes the ability toautomate the process. Seventh, the technique has low sensitivity andgenerally is only effective with the contents of hundreds of thousandsor millions of cells. Certainly, the technique cannot be used to analyzethe enzymes of an individual mammalian cell. Eighth, the quantitation ofthe technique is poor. For one example, the agitation process may failto cause the uniform binding of reporter substrates to the analytes inthe blotting material. For another example, in the electroblotting stepthe time required to transfer some proteins is sufficient to enableother proteins to pass through the blotting membrane and be lost.Finally, the process can require large quantities of expensive probe andreporter antibodies to be used. In sum, the Western gel blottingtechnique is generally complex, time-consuming, expensive, insensitiveand inexact.

Thus, although numerous approaches have been developed for detectinganalytes, there is still a great need to find new methods and devicesthat can be used to conveniently and sensitively detect and characterizea wide variety of analytes.

4. SUMMARY

The present invention provides methods, devices, and kits for detectingone or more analytes of interest in a sample. In some embodiments,methods of detecting at least one analyte in a sample are provided,characterized in that: one or more analytes are resolved in a fluid pathand the analyte(s) are immobilized in the fluid path. Detection agentsare conveyed through the fluid path which bind to or interact with theanalyte(s) and permit detection of the immobilized analyte(s) in thefluid path.

In another aspect, methods for detecting at least one protein in asample are provided comprising the steps of: resolving one or moreproteins in a capillary, photoimmobilizing one or more proteins in thecapillary, contacting antibodies with the immobilized protein(s) to formantibody-protein complex(es) in the capillary, and detecting theprotein(s).

In a further aspect, methods of detecting at least one protein in asample are provided wherein one or more target proteins are resolved ina capillary. The capillary comprises at least one or more photoreactivegroups. In some embodiments, the capillary comprises polymeric materialor polymerizable material comprising one or more photoreactive groups.The protein(s) are photoimmobilized in the capillary. Antibodies arethen contacted with the photoimmobilized proteins and formantibody-protein complex(es) in the capillary, and the proteins aredetected.

Further methods of detecting at least one protein in a sample areprovided comprising the steps of: concentrating one or more proteins ina fluid path, immobilizing the protein(s) in the fluid path; contactingthe immobilized target protein(s) with detection agents to form adetection agent-protein complex(es) in the fluid path, and detecting thetarget protein.

Additionally, systems for detecting at least one analyte in a sample areprovided, comprising a fluid path with one or more reactive groupscontained therein, where the reactive groups are capable of immobilizingthe analyte(s) in the fluid path. A power supply is coupled to the fluidpath and is configured to apply a voltage along the fluid path whereinthe analytes are resolved in the fluid path. A detector is providedwhich detects the analytes immobilized in the fluid path.

In another aspect, kits for detecting at least one analyte in a sampleare provided, comprising one or more fluid paths comprising one or morereactive moieties, buffer and detection agents.

These and other features of the present teachings are set forth below.

5. BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIGS. 1 a-d illustrate exemplary embodiments of resolving, immobilizingand labeling cellular materials in a capillary.

FIGS. 2 a-b illustrate exemplary embodiments of immobilizing resolvedanalytes in a polymeric material in a capillary.

FIGS. 3 a-h illustrate exemplary embodiments of detecting one or moreanalytes.

FIG. 4 illustrates an exemplary embodiment of detecting cellularmaterials.

FIG. 5 illustrates an exemplary embodiment of analyzing cell(s).

FIG. 6 illustrates an exemplary embodiment of detecting cellularmaterials.

FIG. 7 illustrates an exemplary embodiment of analyzing cell(s).

FIG. 8 illustrates an exemplary embodiment of analyzing cell(s).

FIG. 9 illustrates an exemplary embodiment of analyzing cell(s).

FIG. 10 illustrates an exemplary embodiment of analyzing cellularmaterials.

FIG. 11 illustrates an exemplary embodiment of method for analyzingcellular materials.

FIGS. 12 a-b illustrate exemplary embodiments of (a) a capillary betweentwo fluid-filled wells and electrodes and (b) a capillary array device.

FIG. 13 illustrates an exemplary embodiment of an analytical systemdetection of cellular materials in a capillary by chemiluminescence.

FIG. 14 illustrates an exemplary embodiment of an analytical device.

FIG. 15 illustrates an exemplary embodiment of an analytical device.

FIG. 16 illustrates an exemplary embodiment of an analytical device.

FIG. 17 illustrates fluorescent detection of Green Fluorescent Protein.

FIG. 18 illustrates chemiluminescent detection of Green FluorescentProtein.

FIG. 19 illustrates fluorescent detection of horse myoglobin.

FIG. 20 illustrates chemiluminescent detection of Akt protein.

FIG. 21 illustrates chemiluminescent detection of phosphorylated Aktprotein.

FIG. 22 illustrates chemiluminescence detection of Akt protein andphosphorylated Akt protein.

6. DETAILED DESCRIPTION OF EMBODIMENTS

It is to be understood that both the foregoing general description andthe following description are exemplary and explanatory only and are notrestrictive of the methods and devices described herein. In thisapplication, the use of the singular includes the plural unlessspecifically state otherwise. Also, the use of “or” means “and/or”unless state otherwise. Similarly, “comprise,” “comprises,”“comprising,” “include,” “includes” and “including” are not intended tobe limiting.

6.1 Definitions

As used throughout the instant application, the following terms shallhave the following meanings:

“Antibody” has its standard meaning and is intended to refer tofull-length as well antibody fragments, as are known in the art,including Fab, Fab₂, single chain antibodies (Fv for example),monoclonal, polyclonal, chimeric antibodies, etc., either produced bythe modification of whole antibodies or those synthesized de novo usingrecombinant DNA technologies.

“Detect” and “detection” have their standard meaning, and are intendedto encompass detection including the presence or absence, measurement,and/or characterization of an analyte.

“Label” as used herein refers to a detectable moiety. As will beappreciated by those in the art, suitable labels encompass a widevariety of possible moieties. In general, labels include, but are notlimited to, a) isotopic labels, which may be radioactive or heavyisotopes; b) immune labels, which may be antibodies or antigens; c)optical dyes, including colored or fluorescent dyes; d) enzymes such asalkaline phosphatase and horseradish peroxidase, e) particles such ascolloids, magnetic particles, etc., and combinations thereof such asfluorescent labeled antibodies, and chemiluminescent labeled antibodies.

“Protein” has its standard meaning and is intended to refer to proteins,oligopeptides and peptides, derivatives and analogs, including proteinscontaining non-naturally occurring amino acids and amino acid analogs,and peptidomimetic structures, and includes proteins made usingrecombinant techniques, i.e. through the expression of a recombinantnucleic acid.

6.2 Methods

Provided herein are methods of detecting one or more analytes in asample. In some embodiments, methods of detecting at least one analytein a sample are provided, characterized in that: one or more analytesare resolved in a fluid path and the analyte(s) are immobilized in thefluid path. Detection agents are conveyed through the fluid path, whichbind to or interact with the analytes and permit detection of theimmobilized analytes in the fluid path.

In some embodiments, methods of detecting at least one analyte ofinterest in a sample are provided. In some embodiments, the methodcomprises resolving one or more analytes in a fluid path, immobilizingthe analytes in the fluid path, and contacting the immobilized analyteswith detection agents, and detecting the analytes. In some embodiments,the method comprises separating a sample into two or more components ina fluid path, immobilizing one or more analytes of interest in the fluidpath, and contacting the immobilized analytes with detection agents, anddetecting the analytes.

The sample contains the analyte or analytes to be detected. The samplecan be heterogeneous, containing a variety of components, i.e. differentproteins. Alternatively, the sample can be homogenous, containing onecomponent. The sample can be naturally occurring, a biological material,or man-made material. For example, the sample can be a single cell or aplurality of cells, a blood sample, a tissue sample, a skin sample, aurine sample, a water sample, or a soil sample. In some embodiments, thesample comprises the contents of a single cell, or the contents of aplurality of cells. The sample can be from a living organism, such as aeukaryote, prokaryote, mammal, human, yeast, or bacterium, or the samplecan be from a virus. In some embodiments, sequential samples can beassayed from a single animal, such as sequential cuttings from a rodenttail over time.

In some embodiments, the sample can be one or more stem cells. A stemcell is any cell that has the ability to divide for indefinite periodsof time and to give rise to specialized cells. Suitable examples includeembryonic stem cells, such as human embryonic stem cells (hES), andnon-embryonic stems cells, such as mesenchymal, hematopoietic, or adultstem cells (MSC).

As will be appreciated by those skilled in the art, virtually anyprocessing may be performed on the sample prior to detecting theanalyte. For example, the sample can be subjected to a lysing step,denaturation step, heating step, purification step, precipitation step,immunoprecipitation step, column chromatography step, centrifugation,etc. In some embodiments, the separation of the sample andimmobilization may be performed on native substrates, the analyte ofinterest, i.e. a protein, or may also undergo denaturation to exposetheir internal hydrophobic groups for immobilizing in the fluid path.

The analyte to be detected can be any analyte selected by the user. Theanalyte can comprise any organic or inorganic molecule capable of beingdetected. Non-limiting examples of analytes that can be detected includeproteins, oligopeptides and peptides, derivatives and analogs, includingproteins containing non-naturally occurring amino acids and amino acidanalogs. Other example of analytes that can be detected includecarbohydrates, polysaccharides, glycoproteins, viruses, metabolites,cofactors, nucleotides, polynucleotides, transition state analogs,inhibitors, drugs, nutrients, electrolytes, hormones, growth factors andother biomolecules as well as non-biomolecules, as well as fragments andcombinations of all the forgoing.

As will be appreciated by those in the art, virtually any method ofloading the sample in the fluid path may be performed. For example, thesample can be loaded into one end of the fluid path. In someembodiments, the sample is loaded into one end of the fluid path byhydrodynamic flow. For example, in embodiments wherein the fluid path isa capillary, the sample can be loaded into one end of the capillary byhydrodynamic flow, such that the capillary is used as a micropipette.FIG. 3 b illustrates an exemplary embodiment of loading a sample incapillary by capillary action. In some embodiments, the sample can beloaded into the fluid path by electrophoresis, for example, when thefluid path is gel filled and therefore more resistant to hydrodynamicflow.

The fluid path can comprise any structure that allows liquid ordissolved molecules to flow. Thus, the fluid path can comprise anystructure known in the art, so long as it is compatible with the methodsand devices described herein. In some embodiments, the fluid path is abore or channel through which a liquid or dissolved molecule can flow.In some embodiments, the fluid path is passage in a permeable materialin which liquids or dissolved molecules can flow.

The fluid path comprises any material that allows the detection of theanalyte within the fluid path. The fluid path comprises any convenientmaterial, such as glass, plastic, silicon, fused silica, gel, or thelike. In some embodiments, the method employs a plurality of fluidpaths. A plurality of fluid paths enables multiple samples to beanalyzed simultaneously.

The fluid path can vary as to dimensions, width, depth andcross-section, as well as shape, being rounded, trapezoidal,rectangular, etc., for example. The fluid path can be straight, rounded,serpentine, or the like. As described below, the length of the fluidpath depends in part on factors such as sample size and the extent ofsample separation required to resolve the analyte or analytes ofinterest.

In some embodiments, the fluid path comprises a tube with a bore, suchas a capillary. In some embodiments, the method employs a plurality ofcapillaries. Suitable sizes include, but are not limited to, capillarieshaving internal diameters of about 10 to about 1000 μm, although moretypically capillaries having internal diameters of about 25 to about 400μm can be utilized. Smaller diameter capillaries use relatively lowsample loads while the use of relatively large bore capillaries allowsrelatively high sample loads and can result in improved signaldetection.

The capillaries can have varying lengths. Suitable lengths include, butare not limited to, capillaries of about 2 to 20 cm in length, althoughsomewhat shorter and longer capillaries can be used. In someembodiments, the capillary is about 3, 4, 5, or 6 cms in length. Longercapillaries typically result in better separations and improvedresolution of complex mixtures. Longer capillaries can be of particularuse in resolving low abundance analytes.

Generally, the capillaries are composed of fused silica, althoughplastic capillaries and PYREX (i.e., amorphous glass) can be utilized.As noted above, the capillaries do not need to have a round or tubularshape, other shapes, so long as it is compatible with the methods anddevices described herein can also be utilized.

In some embodiments, the fluid path can be a channel. In someembodiments, the method employs a plurality of channels. In someembodiments, the fluid path can be a channel in a microfluidic device.Microfluidics employs channels in a substrate to perform a wide varietyof operations. The microfluidic devices can comprise one or a pluralityof channels contoured into a surface of a substrate. The microfluidicdevice can be obtained from a solid inert substrate, and in someembodiments in the form of a chip. The dimensions of the microfluidicdevice are not critical, but in some embodiments the dimensions are inthe order of about 100 μm to about 5mm thick and approximately about 1centimeters to about 20 centimeters on a side. Suitable sizes include,but are not limited to, channels having a depth of about 5 μm to about200 μm, although more typically having a depth of about 20 μm to about100 μm can be utilized. Smaller channels, such as micro or nanochannelscan also be used, so long as it is compatible with the methods anddevices described herein.

In some embodiments, the fluid path comprises a gel. In someembodiments, the gel is capable of separating the components of thesample based on molecular weight. A wide variety of such gels are knownin the art, a non-limiting example includes a polyacrylamide gel.

The methods generally comprise resolving one or more analytes, containedin a sample, in the fluid path. Methods of separating a mixture into twoor more components are well know to those of ordinary skill in the art,and may include, but are not limited to, various kinds ofelectrophoresis. As used herein, electrophoresis refers to the movementof suspended or dissolved molecules through a fluid or gel under theaction of an electromotive force applied to electrodes in contact with afluid.

In some embodiments, resolving one or more analytes comprisesisoelectric focusing (IEF) of a sample. In an electric field, a moleculewill migrate towards the pole (cathode or anode) that carries a chargeopposite to the net charge carried by the molecule. This net chargedepends in part on the pH of the medium in which the molecule ismigrating. One common electrophoretic procedure is to establishsolutions having different pH values at each end of an electric field,with a gradient range of pH in between. At a certain pH, the isoelectricpoint of a molecule is obtained and the molecule carries no net charge.As the molecule crosses the pH gradient, it reaches a spot where its netcharge is zero (i.e., its isoelectric point) and it is thereafterimmobile in the electric field. Thus, this electrophoresis procedureseparates molecules according to their different isoelectric points.

In some embodiments, for example when resolving is by isoelectricfocusing, an ampholyte reagent can be loaded into the fluid path. Anampholyte reagent is a mixture of 1 0 molecules having a range ofdifferent isoelectric points. Typical ampholyte reagents are Pharmalyte™and Ampholine™ available from Amersham Biosciences of Buckinghamshire,England. Ampholytes can be supplied at either end of the fluid path, orboth, by pumping, capillary action, gravity flow, electroendosmoticpumping, or electrophoresis, or by gravity siphon that can extendcontinuously through the fluid path.

In some embodiments, resolving one or more analytes compriseselectrophoresis of a sample in a polymeric gel. Electrophoresis in apolymeric gel, such as a polyacrylamide gel or an agarose gel separatesmolecules on the basis of the molecule's size. A polymeric gel providesa porous passageway through which the molecules can travel. Polymericgels permit the separation of molecules by molecular size because largermolecules will travel more slowly through the gel than smallermolecules.

In some embodiments, resolving one or more analytes comprises micellarelectrokinetic chromatography (MEKC) of a sample. In micellarelectrokinetic chromatography, ionic surfactants are added to the sampleto form micelles. Micelles have a structure in which the hydrophobicmoieties of the surfactant are in the interior and the charged moietiesare on the exterior. The separation of analyte molecules is based on theinteraction of these solutes with the micelles. The stronger theinteraction, the longer the solutes migrate with the micelle. Theselectivity of MEKC can be controlled by the choice of surfactant andalso by the addition of modifiers to the sample. Micellar electrokineticchromatography allows the separation of neutral molecules as well ascharged molecules.

The methods comprise immobilizing one or more resolved analytes in thefluid path. As used herein, immobilizing refers to substantiallyreducing or eliminating the motion of molecules in the fluid path. Theimmobilization can be via covalent bonds or non-covalent means such asby hydrophobic or ionic interaction. In some embodiments, the resolvedanalytes of the sample are immobilized in the fluid path by isoelectricfocusing.

In some embodiments, the fluid path comprises one or more reactivemoieties. A reactive moiety can be used to covalently immobilize theresolved analyte or analytes in the fluid path. The reactive moiety cancomprise any reactive group that is capable of forming a covalentlinkage with a corresponding reactive group of individual molecules ofthe sample. Thus, the reactive moiety can comprise any reactive groupknown in the art, so long as it is compatible with the methods anddevices described herein. In some embodiments, the reactive moietycomprises a reactive group that is capable of forming a covalent linkagewith a corresponding reactive group of an analyte of interest. Inembodiments employing two or more reactive moieties, each reactivemoiety can be the same, or some or all of the reactive moieties maydiffer.

The reactive moiety can be attached directly, or indirectly to the fluidpath. In some embodiments, the reactive moiety can be supplied insolution or suspension, and may form bridges between the wall of thefluid path and the molecules in the sample upon activation. The reactivemoiety can line the fluid path or, in another embodiment, may be presenton a linear or cross-linked polymer in the fluid path. The polymer mayor may not be linked to the wall of the fluid path before and/or afteractivation.

A wide variety of reactive moieties suitable for covalently linking twomolecules together are well-known. The actual choice of reactivemoieties will depend upon a variety of factors, and will be apparent tothose of skill in the art. For example, the reactive moiety can bind tocarbon-hydrogen (C-H) bonds of proteins. Since many separation mediaalso contain components with C-H bonds, chemistries that react withsulfhydryl (S-H) groups may be advantageous in that S-H groups are founduniquely on proteins relative to most separation media components.Chemistries that react with amine or carboxyl groups may also beadvantageous due to the prevalence of such groups on proteins.

Suitable reactive moieties include, but are not limited to,photoreactive groups, chemical reactive groups, and thermoreactivegroups.

Photoimmobilization in the fluid path can be accomplished by theactivation of one or more photoreactive groups. A photoreactive groupcomprises one or more latent photoreactive groups that upon activationby an external energy source, forms a covalent bond with othermolecules. See, e.g., U.S. Pat. Nos. 5,002,582 and 6,254,634, thedisclosures of which are incorporated herein by reference. Thephotoreactive groups generate active species such as free radicals andparticularly nitrenes, carbenes, and excited states of ketones uponabsorption of electromagnetic energy. The photoreactive groups can bechosen that are responsive to various portions of the electromagneticspectrum, such as those responsive to ultraviolet, infrared and visibleportions of the spectrum. For example, upon exposure to a light source,the photoreactive group can be activated to form a covalent bond with anadjacent molecule.

Suitable photoreactive groups include, but are not limited to, arylketones, azides, diazos, diazirines, and quinones.

In some embodiments, the photoreactive group comprises aryl ketones,such as benzophenone, acetophenone, anthraquinone, anthrone, andanthrone-like heterocycles or their substituted derivatives.Benzophenone is a preferred photoreactive moiety, since it is capable ofphotochemical excitation with the initial formation of an excitedsinglet state that undergoes intersystem crossing to the triplet state.The excited triplet state can insert into carbon-hydrogen bonds byabstraction of a hydrogen atom to create a radical pair. The subsequentcollapse of the radical pair leads to formation of a new carbon-carbonbond. If a reactive bond (e.g., carbon-hydrogen) is not available forbonding, the ultraviolet light-induced excitation of the benzophenonegroup is reversible and the molecule returns to ground state energylevel upon removal of the energy source.

In some embodiments, the photoreactive group comprises azides, such asarylazides such as phenyl azide, 4-fluoro-3-nitrophenyl azide, acylazides such as benzoyl azide and p-methylbenzoyl azide, azido formatessuch as ethyl azidoformate, phenyl azidoformate, sulfonyl azides such asbenzenesulfonyl azide, and phosphoryl azides such as diphenyl phosphorylazide and diethyl phosphoryl azide.

In some embodiments, the photoreactive group comprises diazo compoundsand includes diazoalkanes such as diazomethane and diphenyldiazomethane,diazoketones such as diazoacetophenone and1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates such as t-butyldiazoacetate and phenyl diazoacetate, and beta-keto-alpha-diazoacetatessuch as t-butyl alpha diazoacetoacetate.

In some embodiments, the photoreactive group comprises diazirines suchas 3-trifluoromethyl-3-phenyldiazirine, and photoreactive groupcomprises ketenes such diphenylketene.

In some emdodiments, the photoreactive group comprises aN-((2-pyridyldithio)ethyl)-4-azidosalicylamide,4-azido-2,3,5,6-tetrafluorobenzoic acid,4-azido-2,3,5,6-tetrafluorobenzyl amine, benzophenone-4-maleimide,benzophenone-4-isothiocyanate, or 4-benzoylbenzoic acid.

As described above, in embodiments employing two or more reactivemoieties, each reactive moiety can be the same, or some or all of thereactive moieties may differ. For example, the fluid path can comprisephotoreactive groups and chemically reactive. In some embodiments, thefluid path can comprise different photoreactive groups, non limitingexamples include, benzophenone and 4-azido-2,3,5,6-tetrafluorobenzoicacid (ATFB).

In addition to the use of photoactivatable chemistry described above,chemical or thermal activation may also be employed.

In some embodiments, the reactive moiety comprises a functional groupthat can be used to attach the reactive moiety to an analyte by forminga covalent linkage with a complementary group present on the analyte.Pairs of complementary groups capable of forming covalent linkages arewell known in the art. In some embodiments, the analyte comprises anucleophilic group and the reactive group comprises an electrophilicgroup. In other embodiments, the reactive group comprises a nucleophilicgroup and the analyte comprises an electrophilic group. Complementarynucleophilic and electrophilic groups, or precursors thereof that can besuitably activated, useful for forming covalent linkages stable in assayconditions are well known and can be used. Examples of suitablecomplementary nucleophilic and electrophilic groups, as well as theresultant linkages formed there from, are provided in U.S. Pat. No.6,348,596.

In some embodiments, the methods comprise contacting one or moreanalytes with one or more detection agents. A detection agent is capableof binding to or interacting with the analyte be detected. Contactingthe detection agent with the analyte or analytes of interest can be byany method known in the art, so long as it is compatible with themethods and devices described herein. Examples for conveying detectionagents through the fluid path include, but are not limited to,hydrodymic flow, electroendosmotic flow, or electrophoresis.

The detection agents can comprise any organic or inorganic moleculecapable of binding to interact with the analyte to be detected.Non-limiting examples of detection agents include proteins, peptides,antibodies, enzyme substrates, transition state analogs, cofactors,nucleotides, polynucleotides, aptamers, lectins, small molecules,ligands, inhibitors, drugs, and other biomolecules as well asnon-biomolecules capable of binding the analyte to be detected.

In some embodiments, the detection agents comprise one or more labelmoiety(ies). In embodiments employing two or more label moieties, eachlabel moiety can be the same, or some, or all, of the label moieties maydiffer.

In some embodiments, the label moiety comprises a chemiluminescentlabel. The chemiluminescent label can comprise any entity that providesa light signal and that can be used in accordance with the methods anddevices described herein. A wide variety of such chemiluminescent labelsare known in the art. See, e.g., U.S. Pat. Nos. 6,689,576, 6,395,503,6,087,188, 6,287,767, 6,165,800, and 6,126,870 the disclosures of whichare incorporated herein by reference. Suitable labels include enzymescapable of reacting with a chemiluminescent substrate in such a way thatphoton emission by chemiluminescence is induced. Such enzymes inducechemiluminescence in other molecules through enzymatic activity. Suchenzymes may include peroxidase, beta-galactosidase, phosphatase, orothers for which a chemiluminescent substrate is available. In someembodiments, the chemiluminescent label can be selected from any of avariety of classes of luminol label, an isoluminol label, etc. In someembodiments, the detection agents comprise chemiluminescent labeledantibodies.

In some embodiments, the detection agents comprise chemiluminescentsubstrates. Depending on their charge, the chemiluminescent substratescan be supplied from either end of the fluid path, once the analyte isimmobilized in the fluid path. Uncharged substrates can be supplied fromeither end of the fluid path by hydrodynamic flow or electroendosmoticflow, for example. Chemiluminescent substrates are well known in theart, such as Galacton substrate available from Applied Biosystems ofFoster City, Calif. or SuperSignal West Femto Maximum Sensitivitysubstrate available from Pierce Biotechnology, Inc. of Rockford, Ill. orother suitable substrates.

Likewise, the label moiety can comprise a bioluminescent compound.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent compound isdetermined by detecting the presence of luminescence. Suitablebioluminescent compounds include, but are not limited to luciferin,luciferase and aequorin.

In some embodiments, the label moiety comprises a fluorescent dye. Thefluorescent dye can comprise any entity that provides a fluorescentsignal and that can be used in accordance with the methods and devicesdescribed herein. Typically, the fluorescent dye comprises aresonance-delocalized system or aromatic ring system that absorbs lightat a first wavelength and emits fluorescent light at a second wavelengthin response to the absorption event. A wide variety of such fluorescentdye molecules are known in the art. For example, fluorescent dyes can beselected from any of a variety of classes of fluorescent compounds,non-limiting examples include xanthenes, rhodamines, fluoresceins,cyanines, phthalocyanines, squaraines, bodipy dyes, coumarins, oxazines,and carbopyronines. In some embodiments, for example, where detectionagents contain fluorophores, such as fluorescent dyes, theirfluorescence is detected by exciting them with an appropriate lightsource, and monitoring their fluorescence by a detector sensitive tothere characteristic fluorescence emission wavelength. In someembodiments, the detection agents comprise fluorescent dye labeledantibodies.

In embodiments, using two or more different detection agents, which bindto or interact with different analytes, different types of analytes canbe detected simultaneously. In some embodiments, two or more differentdetection agents, which bind to or interact with the one analyte, can bedetected simultaneously. In embodiments, using two or more differentdetection agents, one detection agent, for example a 1° antibody, canbind to or interact with one or more analytes to form a detectionagent-analyte complex, and second detection agent, for example a 2°antibody, can be used to bind to or interact with the detectionagent-analyte complex.

In some embodiments, two different detection agents, for exampleantibodies for both phospho- and non-phospho- forms of analyte ofinterest can enable detection of both forms of the analyte of interest.In some embodiments, a single specific detection agent, for example anantibody, can allow detection and analysis of both phosphorylated andnon-phosphorylated forms of a analyte, as these can be resolved in thefluid path. In some embodiments, multiple detection agents can be usedwith multiple substrates to provide color-multiplexing. For example, thedifferent chemiluminescent substrates used would be selected such thatthey emit photons of differing color. Selective detection of differentcolors, as accomplished by using a diffraction grating, prism, series ofcolored filters, or other means allow determination of which colorphotons are being emitted at any position along the fluid path, andtherefore determination of which detection agents are present at eachemitting location. In some embodiments, different chemiluminescentreagents can be supplied sequentially, allowing different bounddetection agents to be detected sequentially.

Analyte detection includes detecting of the presence or absence,measurement, and/or characterization of an analyte. Typically, ananalyte is detected by detecting a signal from a label and includes, butis not limited, to detecting isotopic labels, immune labels, opticaldyes, enzymes, particles and combinations thereof such aschemiluminescent labeled antibodies and fluorescent labeled antibodies.

Detecting the analyte can be by any method known in the art, so long asit is compatible with the methods and devices described herein. Analytedetection can be performed by monitoring a signal using conventionalmethods and instruments, non-limiting examples include, a photodetector,an array of photodetectors, a charged coupled device (CCD) array, etc.For example, a signal can be a continuously monitored, in real time, toallow the user to rapidly determine whether an analyte is present in thesample, and optionally, the amount or activity of the analyte. In someembodiments, the signal can be measured from at least two different timepoints. In some embodiments, the signal can be monitored continuously orat several selected time points. Alternatively, the signal can bemeasured in an end-point embodiment in which a signal is measured aftera certain amount of time, and the signal is compared against a controlsignal (sample without analyte), threshold signal, or standard curve.

A signal can be a monitored, in real time, to allow the user to rapidlydetermine whether an analyte is present in the sample, and optionally,the amount or activity of the analyte. In some embodiments, the signalcan be measured from at least two different time points. In someembodiments, the signal can be monitored continuously or at severalselected time points. Alternatively, the signal can be measured in anend-point embodiment in which a signal is measured after a certainamount of time, and the signal is compared against a control signal(sample without analyte), threshold signal, or standard curve.

Typically, detecting the analyte comprises imaging the fluid path. Insome embodiments, the entire length of the fluid path can be imaged.Alternatively, a distinct part or portion of the fluid path can beimaged. The amount of the signal generated is not critical and can varyover a broad range. The only requirement is that the signal bemeasurable by the detection system being used. In some embodiments, asignal can be at least 2-fold greater than the background. In someembodiments, a signal between 2 to 10-fold greater than the backgroundcan be generated. In some embodiments, a signal can be 10-fold greaterthan the background.

The amount of the signal generated is not critical and can vary over abroad range. The only requirement is that the signal be measurable bythe detection system being used. In some embodiments, a signal can be atleast 2-fold greater than the background. In some embodiments, a signalbetween 2 to 10-fold greater than the background can be generated. Insome embodiments, a signal can be 10-fold greater than the background.

FIG. 1 a-d illustrate exemplary embodiments of resolving, immobilizingand labeling cellular materials in a capillary. FIG. 1 a is alongitudinal cross-sectional illustration of a capillary 10 which islined with a photoreactive group 12. Located within a fluid inside thecapillary is a mixture of cellular proteins 14 of differingelectrophoretic mobility as indicated by the different shading. In FIG.1 b an electric field has been applied to the fluid to separate theproteins in accordance with their isoelectric points by isoelectricfocusing (IEF) into groups 14 a, 14 b, and 14 c. In FIG. 1 c light 15 atthe appropriate wavelength is applied to activate the photoreactivegroup which, when activated as indicated at 12a, binds the proteins 14at their separated locations within the capillary. Detection antibodies16 carrying a label are then flowed through the capillary as indicatedby arrow 18 in FIG. Id. The detection antibodies 16 will bind to theproteins 14 they encounter as shown in FIG. 1 d. When the detectionantibodies contain chemiluminescent label, the bound proteins are thenlabeled in their bound locations for luminescent detection. In thisembodiment, a stream of chemiluminescence reagents can be flowed throughthe capillary, reacting when encountering the label linked to theproteins. The luminescence from the sites of the proteins is detected bya photon detector and recorded, enabling identification of the proteinsby the light emitted from their bound locations. The techniqueadvantageously permits the identification of cellular materials and, inthe case where modification of cellular materials (substrates) is beingmonitored, allows the use of these native substrates without the need tointroduce any identification substances prior to the separation of thecellular materials by IEF.

Generally, the methods described herein yield results similar to thoseobtained by a Western blot but in a fraction of the time. For example,the separation of cellular materials by IEF can take 5 minutes or less,and subsequent immobilization takes 2 minutes or less. This means thatthe detection agents can be linked to the separated sample within 10minutes or less of the commencement of separation, and that thedetection agents can be analyzed within 30 minutes of the separatingstep. The entire process is faster, simpler, more sensitive, moreaccurate and more automatable than the Western blot analyticaltechnique. The immobilization step obviates the need to assess thedetection agents (such as enzyme-labeled antibodies) for homogeneity ofmolecular form prior to use and obviates the need for excessivepurification not typical of these types of reagents. Thus, less costlyprobing antibodies can be used in the methods described herein.

While the separation technique shown in the previous embodiment isisoelectric focusing, free solution electrophoresis, sievingelectrophoresis, or micellar electrokinetic chromatography for example,may also be used to resolve the analytes.

In some embodiments, methods of detecting at least one analyte isprovided, comprising, resolving one or more proteins in a fluid path,immobilizing the analytes in the fluid path; and contacting theimmobilized analytes with detection agents to form one or more detectionagent-analyte complexes in the fluid path, and detecting the analyte. Insome embodiments, the detection agent comprises a label. In someembodiments, the method further comprises contacting the detectionagent-analyte complex with a labeled detection agent. In someembodiments, the method comprises detecting a chemiluminescent signal.In some embodiments, the method comprises detecting a fluorescentsignal.

In some embodiments, methods of detecting at least one protein ofinterest in a sample are provided, comprising: resolving one or moreproteins in a capillary, photoimmobilizing the proteins in thecapillary, and contacting the immobilized proteins with antibodies toform one or more antibody-protein complexes in the capillary anddetecting the protein. In some embodiments, the antibodies comprise alabel. In some embodiments, the method further comprising contacting theantibody- protein complex with a labeled antibody. In some embodiments,the method comprises detecting a chemiluminescent signal. In someembodiments, the method comprises detecting a fluorescent signal.

In some embodiments, methods of detecting at least one analyte in asample are provided, comprising: resolving one or more analytes in afluid path, wherein the fluid path comprises one or more reactive groupsand, optionally, polymeric or polymerizable materials comprising one ormore reactive groups, and immobilizing the analytes in the fluid pathand contacting detection agents to the immobilized analytes anddetecting the analytes.

FIG. 2 a-b illustrate exemplary embodiments of immobilizing resolvedanalytes, in a polymeric material in a capillary. FIG. 2 a illustrates alongitudinal cross section of a capillary 10. The upper panel showscapillary 10 walls coated on their interior surface with a photoreactivegroup 12, represented by closed ovals. A suitable, non-limiting exampleof such material is polyacrylamide containing photoreactive groups, suchas benzophenone moieties. Also present in FIG. 2 a are polymericmaterials 24 in solution, represented by four-armed structuresterminated in circles, where the circles represent photoreactive groups12. A suitable, non-limiting example of such material is branchedpolyethylene glycol bearing photoreactive groups 12 such asbenzophenone, ATFB etc. In addition, two bands of resolved proteins 14a, 14 b are shown, represented by the cross-hatched structures. FIG. 2 bshows the structures described above after photoactivation 15.Activation of the photoreactive groups is depicted by the concavesemicircular structures 12 a on both the walls and the polymericmaterials filling the capillary. Many of these photoreactive groups 12 aare associated with each other, with lengths of polymer, and with theproteins in bands, effectively cross-linking each of these together.Thus, the resolved protein bands are bound in place via a loose networkof covalent bonds and polymeric materials. In some embodiments, it isdesirable that the network form open-pored structures permitting themovement of materials such as detection agents, such as antibodies,through the loose network.

FIGS. 3 a-h illustrate exemplary embodiments for detecting one or moreanalytes in a fluid path. FIG. 3 a illustrates a capillary 10 and asample 1 comprising a mixture of components containing one or moreanalytes of interest. FIG. 3 b illustrates loading the sample 1 into thecapillary 10 by capillary action. FIG. 3 c illustrates the sample 1loaded capillary 10, comprising one or more reactive moieties, extendingbetween two fluid-filled wells or troughs 20 a, 20 b. The components ofthe sample 1 are separated such that the analyte 1 a or analytes 1 a and1 b of interests are resolved by one or more electrodes in contact witha solution on one side of the capillary 10 and another one or moreelectrodes is in contact with a solution on the other side of thecapillary 10 as illustrated in FIG. 3 d. FIG. 3 e illustrates theactivation of one or more reactive moieties capable of immobilizing theanalytes 1 a and 1 b of interests in the capillary 10. Detection agents2 are then flowed through the capillary 10 as indicated by the arrow inFIGS. 3 f and 3 g. Detection agents 2 are then detected 3, enablingdetection of the analyte of interest in their immobilized locations inthe capillary by the signal emitted as illustrated in FIG. 3 h.

FIG. 4 illustrates an exemplary embodiment of method for analyzingcellular materials. In step 61 the cellular materials to be analyzed arelocated at one end of a capillary. In step 61a the cellular materialsare loaded in to the capillary. In step 62 the cellular materials areseparated within the capillary, for example by IEF. In step 63 theseparated materials are immobilized in the capillary. In step 64detection agents, for example reporter antibodies, are bound to theimmobilized analytes, such as proteins in the capillary. In step 65 achemiluminescent reagents, or other detection agents, are flowed throughthe capillary to produce the event to be detected, such aschemiluminescence. The emitted light is then detected in step 66.

FIG. 5 illustrates an exemplary embodiment of a method of analyzingcell(s). In step 60 one or more cells to be analyzed are positioned atthe end of a capillary. In step 60 a one or more cells are drawn intothe capillary and are lysed. In step 62 the cellular materials areseparated within the capillary, for example by IEF. In step 63 theseparated materials are immobilized in the capillary. In step 64detection agents, for example reporter antibodies, are bound to theimmobilized analytes, such as proteins in the capillary. In step 65 achemiluminescent reagent, or other detection agents, are flowed throughthe capillary to produce the event to be detected, such aschemiluminescence. The emitted light is then detected in step 66.

FIG. 6 illustrates an exemplary embodiment of a method for analyzingcellular materials. In step 61 the cellular materials to be analyzed arelocated at one end of a capillary. In step 61 a the cellular materialsare loaded into the capillary. In step 62 the cellular materials areseparated within the capillary, for example by IEF. In step 63 theseparated materials are immobilized in the capillary. In step 64detection agents, for example reporter antibodies, are bound to theimmobilized analytes, such as proteins in the capillary. In step 65 afluorophores on the detection agents, for example fluorescent labeledantibodies, are excited with light. The emitted light is then detectedin step 66.

FIG. 7 illustrates an exemplary embodiment of analyzing cells. In step60 one or more cells to be analyzed are positioned at the end of acapillary. In step 60 a one or more cells are drawn into the capillaryand are lysed. In step 62 the cellular materials are separated withinthe capillary, for example proteins are resolved by IEF. In step 63 theseparated materials are immobilized in the capillary. In step 64detection agents, for example reporter antibodies, are bound to theimmobilized analytes, such as proteins in the capillary. In step 65 afluorophores on the detection agents, for example fluorescent labeledantibodies, are exited with light. The emitted light is then detected instep 66.

FIG. 8 illustrates an exemplary embodiment in which labeled cellularmaterials are released from the cell at the moment of their introductioninto the capillary. The cellular materials are then separated andimmobilized. In step 91 one or more cells containing detection agentsare located at one end of a capillary. The cell or cells are then lysedin step 92 to release their labeled proteins and transported in thecapillary. In step 93 the cellular materials are separated, for examplewithin the capillary by IEF. In step 94 the separated materials areimmobilized in the capillary. In step 95 a chemiluminescent reagent isthen flowed through the capillary to produce photons bychemiluminescence. The emitted photons are then detected in step 96.

FIG. 9 illustrates an exemplary embodiment in which analytes are labeledprior to separation. In step 101 one or more cells are located at oneend of a capillary. The cell or cells are then lysed in step 102 torelease their contents. In step 103 detection agents are bound to thereleased cellular contents, for example proteins. In step 104 thecellular materials are separated within the capillary by IEF. In step105 the separated labeled materials are immobilized in place in thecapillary. In step 106 a chemiluminescence substrate is then flowedthrough the capillary to produce photons by chemiluminescence. Theemitted photons are then detected in step 107.

FIG. 10 illustrates an exemplary embodiment for chemiluminescentdetection of analytes. In step 302 an ampholyte reagent for the pHgradient is loaded into the fluid path. In step 304 enzyme-labeledantibodies able to catalyze chemiluminescence and able to bind theanalyte of interest are loaded into the fluid path. The cell contentsare loaded into the fluid path in step 306, whereupon the enzyme-taggedantibodies will bind with the analyte or analytes of interest. Afocusing isoelectric field is applied in step 308 to resolve and thenimmobilize the enzyme-tagged antibodies and analytes in a pH gradient. Achemiluminescent substrate compatible with the enzyme-labeled antibodiesis supplied in step 310 and chemiluminescent emissions are then detectedfrom the interaction of the chemiluminescent substrate with theenzyme-labeled antibodies and bound to analyte in step 312. In someembodiments, the analyte is immobilized by IEF and the chemiluminescentreagent is flowed through the fluid path by carryings it's own charge atall pH's of the gradient.

FIG. 11 illustrates an exemplary embodiment for chemiluminescentdetection of analytes. In this embodiment, a cell is lysed into or atthe inlet of a capillary in step 402. The lysis releases cellularcontents which react with detection agents, for example chemiluminescentlabeled antibodies, in step 404. The labeled and bound cellular contentsare resolved in the capillary by isoelectric focusing in step 406. Instep 408 a chemiluminescent reagent is supplied which will react withthe enzyme of the antibodies bound to the cellular contents. In thisembodiment, the analyte is immobilized by IEF and the chemiluminescentreagent is flown through the fluid path by carryings it's own charge atall pH's of the gradient. In step 410 chemiluminescence is detected witha photon detector such as a photocell or CCD array detector.

Variations of order of the steps of the methods described herein willreadily occur to those skilled in the art. For example, the sample canbe separated and then the analyte(s) immobilized at their resolvedlocations in the fluid path, prior to contacting the analyte(s) with thedetection agents. In some embodiments, detection agents are contactedwith the analyte(s) to form a complex and then the complex is resolvedin the fluid path. In some embodiments, the detection agents could bepreloaded into the sample thereafter loaded into the system. As anotherexample, the resolving step, such as isoelectric focusing can be appliedafter the chemiluminescent reagents are supplied.

Also provided herein are methods of detecting at least one protein in asample, characterized in that: one or more proteins are resolved fromthe sample in a capillary and the proteins are photoimmobilized in thecapillary and antibodies are conveyed through said capillary which bindto or interact with the proteins or an antibody-protein complex andpermit the detecting of the proteins while immobilized in saidcapillary.

Also, provided herein are methods of detecting at least one protein in asample, comprising concentrating one or more analytes in a fluid path,immobilizing one or more analytes in the fluid path; and contacting theimmobilized analyte with detection agents, and detecting the analyte ofinterest.

As used herein, concentrating means to make less dilute. Methods ofconcentrating a sample are well known to those of ordinary skill in theart, and may include, but are not limited to, various kinds ofelectrophoresis and isoelectric focusing etc.

Also provided are methods of detecting at least one protein in a sample,comprising, concentrating one or more proteins in a fluid path,immobilizing the proteins in the fluid path, and contacting theimmobilized protein with antibodies to form one or more antibody-proteincomplexes in the fluid path, and detecting the protein.

6.3 Devices

Provided herein are systems and devices for detecting one or moreanalytes in a sample. The device generally comprises a fluid path; apower supply for applying a voltage along the fluid path for separatingindividual components of a sample in the fluid path; and a detectorcapable of detecting analyte(s) in the fluid path.

Also provided is a system for detecting at least one analyte of interestin a sample, comprising a fluid path comprising one or more reactivegroups capable of immobilizing one or more analytes, a power supply forapplying a voltage along the fluid path capable of resolving one or moreanalytes in the fluid path; and a detector capable of detecting theanalyte(s) in said fluid path.

Also provided is a system for detecting at least one analyte of interestin a sample, comprising a fluid path comprising one or more reactivegroups capable of immobilizing one or more analytes, a power supply forapplying a voltage along the fluid path capable of concentrating one ormore analytes in the fluid path; and a detector capable of detecting theanalyte(s) in said fluid path.

FIG. 12 a illustrates an exemplarily embodiment wherein the fluid pathcomprises a capillary between fluid filled wells and electrodes. Acapillary 10 comprising one or more reactive moieties extends betweentwo fluid-filled wells or troughs 20 a, 20 b. A sample is placed in oneof the troughs, preferably at the orifice of the capillary. For example,the sample can be cellular contents that have been loaded into thecapillary. In some embodiments, the one or more cells are drawn into thecapillary and lysed in-situ. In some embodiments, one or more cells maybe placed in a well, trough or capillary opening and lysed to releasethe cellular contents. The sample is then flowed through the capillaryas by electrophoresis and separated within the capillary, for example byisoelectric focusing. An electrode 22 a, 22 b is located in the solutionat each end of the capillary to apply the electric field necessary forelectrophoresis and isoelectric focusing. The detection agents used tolabel the analyte of interest can be located in the other trough,preferably after separation and immobilization have taken place, andflowed through the capillary by electrophoresis, electroendosmotic flow,or hydrodynamic flow (typically achieved by siphoning or pumping). Insome embodiments, the detection agents can be loaded into the capillaryfrom a vessel, such a test tube. The detection agents are thenintroduced into one of the troughs and flowed through the capillary toelicit the detection events.

FIG. 12 b illustrates an exemplary embodiment comprising an array ofcapillaries 30 extending between a plurality of wells 32 a, on one sideand another plurality of wells 32 b on the other side. In someembodiments an array of capillaries can be extended between a trough, onone side and another trough on the other side. In some emdodiments, anarray of capillaries can extendend between a common buffer reservoir, onone side and another common buffer reservoir on the other side. One ormore electrodes 34 a is in contact with the solution on one side of thecapillaries and another one or more electrodes 34 b is in contact withthe solution on the other side of the capillaries. A portion of theelectrodes may be integral to the reservoir structure. The reservoirsand capillaries are located on or in a substrate 36 such as a slide.

FIG. 13 is an exemplary embodiment, the separation and detection ofantibody-analyte complexes within a capillary 122 is illustrated in alongitudinal cross-sectional view of a section of the capillary. Locatedat positions along the capillary 122 are antibody-analyte complexes 160.Each antibody-analyte complex 160 has a net charge 164 which determinesthe charge-neutral location 162 b to which the complex will migrate.Each complex is located at its charge-neutral location 162 a, 162 b, 162c in the pH gradient created by isoelectric focusing of the ampholytereagent. The applied voltage potential focuses the analytes into narrowbands at these isoelectric locations as illustrated in the drawing.Passing through the capillary are chemiluminescent substrates 170 whichtravel in electrophoretic flow direction 172. When a chemiluminescentsubstrate 170 encounters a labeled antibody-analyte complex 160 such asa peroxidase enzyme attached to an electrofocused antibody-proteincomplex, the chemiluminescent substrate is converted to a product pluslight. The substrate 170 a represents a chemiluminescent substrate whichis being converted. The conversion causes light 180 to be emitted by thesubstrate 170 a. Substrate products 170 b which result from suchconversions continue to flow in the direction of arrow 172. This processcontinues as long as unconverted chemiluminescent substrates 170continue to flow through the capillary and encounter newchemiluminescent enzymes with which to react.

FIG. 14 illustrates an exemplary analytical device. An array ofcapillaries 40 which are loaded with the necessary one or more reactivemoieties to immobilize the analytes, buffer, and sample to be analyzedis located in a light-tight box 42. A controllable power supply 46 iscoupled to the electrodes on either end of the capillaries to apply thevoltages needed to separate the sample and to flow the detection agentsand/or chemiluminescent reagents through the capillaries. A voltage isapplied to flow the sample into the capillary and to separate thesample, for example by isoelectric focusing. Alternatively, the samplemay be loaded into the capillary by hydrodynamic flow and thereafterseparated, for example by isoelectric focusing. An energy source (notshown) capable of activating the reactive moieties is provided. Forexample, a light source such as an ultraviolet lamp providesillumination inside the box to immobilize the individual components ofthe sample in their separated locations. In some embodiments, the systemcomprises a light source for induction of fluorescent label. One or moredetection agents, such as those described herein, are introduced intothe wells at one end of the capillaries and flowed through thecapillaries, binding to the analytes. In some embodiments, detectionreagent is introduced into the wells and flowed through the capillaries.In some embodiments, detection agents may be introduced from separatesmaller wells. Additional smaller wells can be used to conservedectection agents. Viewing the capillaries within the box 42 to receivethe photons emitted from the immobilized analytes and detectionmolecules is a CCD camera 44. The system is controlled by a computer 48which switches the power supply 46 and the light, controls theapplication of detection molecules and reagents, and records andanalyzes the photon signals received by the CCD camera 44. Similarly, alight source for induction of fluorescence of molecular standards run inthe separation may allow detection with the same CCD camera used todetect chemiluminescence-produced light. Internal standards serve tocalibrate the separation with respect to isoelectric point, or for analternative separation mode, molecular weight. Internal standards forIEF are well know in the art, for example see, Shimura, K., Kamiya, K.,Matsumoto, H., and K. Kasai (2002) Fluorescence-Labeled Peptide pIMarkers for Capillary Isoelectric Focusing, Analytical Chemistry v74:1046-1053, and U.S. Pat. No. 5,866,683. Standards to be detected byfluorescence could be illuminated either before or afterchemiluminescence, but generally not at the same time aschemiluminescence.

In some embodiments, the analyte and standards are detected byfluorescence. The analyte and standards can each be labeled withfluorescent dyes that are each detectable at discrete emissionwavelengths, such that the analyte and standards are independentlydetectable.

FIG. 15 is an exemplary embodiment in which the photons emitted from thedetection molecules are received by a CCD array located beneath thecapillary array 40. The CCD array 52 is monitored by a CCD controller 54which provides amplified received signals to the computer 48.

FIG. 16 illustrates an exemplary embodiment of an analytical device forcapillary detection of cellular material by chemiluminescence. Thesystem 110 comprises a microscope 112 having a video ocular readout 114such as a CCD camera displayed on a CRT screen and/or recorded by avideotape recorder or digital recorder (not shown). In some embodiments,the system allows digital storage of the images and pattern processingin a computer system for automated cell processing and analysis. The CCDvideo camera system 114 is capable of recording a real time bright fieldimage of a target cell. The device may optionally comprise a cell lysisdevice 16, such as a laser, sonic generator, electronic pulse generator,or electrodes positioned adjacent to target cell(s) on a cover slip 36.In the illustrated embodiment, after cell lysis, cell contents areloaded into the end of the capillary by hydrodynamic flow orelectrophoresis. In some embodiments, this end of the capillary hasalready been loaded with a short (few mm or less) slug of labeledantibodies at the time of cell lysis. Thereafter, following ahybridization period, if necessary, separation for example byisoelectric focusing is initiated.

A fused silica capillary 122 is positioned with a micromanipulator (notshown) so that the inlet 126 of capillary 122 is located above the coverslip 136 or slide or microwell plate performed by loading the cell withdetection agents prior to lysis or hybridization may be performed in thebuffer solution subsequent to lysis. In the latter event, a highconcentration of detection agents surrounds or is located adjacent tothe cell. One method for achieving the desired high concentration ofcell contents in contact with a high concentration of detection agentsis to draw the cell contents by hydrodynamic or electrophoretic meansinto a short length of the capillary adjacent to the capillary end. Inthis mode this short region of the capillary may be pre-loaded withdetection agents from another source such as a tube or well (not shown),or may be drawn into the capillary end along with the cell contents. Thedistal end 132 or proximal end 126 of capillary 122 is disposed in asolution 134 of chemiluminescent substrates. In some embodiments,resolving and immobilizing the analyte or analytes of interest can occurprior to adding the detection agents. The detection agents are thenflowed though the fluid path after the separating the sample andimmobilization.

Ampholyte reagent 142 is electrically coupled to a high voltagepotential which, when applied to the capillary solution, causes thedevelopment of a pH gradient within the capillary 22 by ampholytemigration. A high voltage power supply, such as model CZE 1000Rmanufactured by Spellman of Plainview, N.Y., which is capable ofproviding a 20,000 volt potential can be used to maintain the pHgradient in column or capillary 122.

Fused silica capillary 122 may typically exhibit a 100 micron innerdiameter and 360 micron outer diameter. The lumen walls can be coatedwith a neutral coating such as that manufactured by Supelco of Phoenix,Ariz. The coating is used to minimize the electroosmotic flow and thusshorten the migration times for the antibody-target complexes. The totallength of the capillary in this embodiment can be as long as 90 to 100cm, but preferably is considerably shorter, in the range of 10 to 30 cm,or 3 to 6 cm. The cell chamber 136 serves as an inlet reservoir fortargeted cell molecules and optionally the ampholyte reagent andchemiluminescent reagent(s) and can be held at ground potential relativeto the high voltage potential at the other end of the capillary. In someembodiments either end of the capillary may be at high voltage potentialand the other ground, or either end may be positive and the othernegative. The outlet reservoir 134 may be held at 15 to 20 kV relativeto ground at the proximal end of the capillary, for example. The actualpotential used is generally chosen by the desired voltage drop per cm ofcapillary. Distal outlet 132 of capillary 122 is placed about 5centimeters below inlet 126 in the case of hydrodynamic loading. For thecase of electrophoretic loading, which may be equally or more effective,no particular elevation of the distal end of the capillary is required.Inlet 126 of capillary 122 is used as a micropipette for introducing thecellular contents into capillary 122 after cell lysis. Alternatively,the cell may be drawn intact into the capillary and then lysed in thecapillary.

After removing 5 mm of polyimide coating from capillary 122 above inlet126, inlet 126 is mounted perpendicular to cover slip 136 by amicromanipulator (not shown). The micromanipulator enables precisepositioning of the capillary lumen with respect to the target cell to beloaded or lysed and loaded into capillary 122.

Capillary 122 includes an optical observation window 138 through whichchemiluminescent or fluorescence events are observed and detected by aCCD array 140 or similar detector. An extended observation window 138 isdesirable as it enables the parallel detection of a greater number ofevents than can be observed through the limited length of a shorterwindow. Generally the length of the observation window will be chosen inconsideration of the length of the CCD array 140 being used. If anon-clear coated capillary is used the polyimide coating of capillary122 is removed over at least the length of the capillary which opposesthe CCD array 140. The observation window 138 is maintained in a fixedposition in relation to the CCD array 140 either by mechanical oradhesive means. Preferably the observation window and CCD array areenclosed in darkness so that the only light detected by the CCD array isthat emitted by the chemiluminescent or fluorescent events within thecapillary. The signals from the detected chemiluminescent or fluorescentevents are coupled to a personal computer 144 where they are recorded.In some embodiments the event data may be recorded along with theposition in the CCD array at which the event occurred. The data isplotted and total signal corresponding to each focused band calculatedusing Origin software available from Microcal of North Hampton, Mass.,DAX software available from Van Mierlo, Inc. of Eindhoven, TheNetherlands, LabVIEW software available from National Instruments Corp.,Austin, Texas, or similar data analysis packages. Data may be presentedas a histogram, electropherogram, or other graphical representation, oras a spreadsheet or other numerical format.

In some embodiments, a cell or cells which have not been preloaded withdetection agents, the inlet 126 of the capillary 122 is positioneddirectly above the target cell or cells to be lysed. The cell or cellscan be in contact with a high concentration of detection agents, orpreferably, a high concentration of detection agents has already beenloaded into the capillary end at the time of cell lysis. The lysisdevice 116 is aimed to create a lysing shock wave or other cell lysingdisruption adjacent to the cell or cells. When the lysing pulse isapplied the cellular contents are released and the force of the lysingevent may aid in propelling the cellular contents into the lumen of thecapillary by hydrodynamic flow, electrophoresis, or electroendosmoticflow. Hybridization of the analytes of interest and the detection agentstakes place rapidly, either outside the capillary prior to loading ofthe cell contents, or inside the capillary. The degree of hybridizationwill be linearly related to the concentrations of the detection agentand the sample. For example, tight-binding (high binding avidity)antibodies provide molecules which will retain their linkingcharacteristics during capillary transport and isoelectric focusing.Examples of such antibodies are those typically used in ELISA (EnzymeLinked Immuno-Sorbent Assays). Preferably the hybridization is doneunder non-denaturing conditions. By causing the antibodies and theiranalytes to be in a natural state, recognition between the antibodiesand their target complexes and the chemiluminescent reporters isenhanced. The isoelectric focusing field is applied, causing theantibody-target complexes to migrate to pH points of the pH gradient inthe capillary at which their net charge is neutral. The complexes willbecome stationary in the capillary at pH points where the charge oftheir molecular components (e.g., phospho, carboxyl, amino, and othercharged functional groups) nets out to zero. If forces from flow ordiffusion should cause the complexes to drift away from their respectiveisoelectric points, the gradient field will migrate them back into theircharge-neutral positions. The antibody-target complexes are thusresolved along the observation window 138 by capillary isoelectricfocusing. In some embodiments, resolving and immobilizing the analyte oranalytes of interest can occur prior to adding the detection agents. Thedetection agents are then flowed though the fluid path after theseparating the sample and immobilization.

The electrophoretic potential is then used to cause the chemiluminescentsubstrate solution 134 to flow through the capillary. This may beinitiated at the same time as the electric field which is first appliedto establish the pH gradient, or after the gradient has already beenestablished and the antibody-target complexes have been focused. Thesubstrate or substrate(s) are chosen such that they exhibit(s) a netcharge at all pH conditions encountered within the capillary so that thesubstrates do not resolve within the capillary but continue to flow in acontinuous stream. As the substrates encounter antibody-target complexesalong the capillary they are cleaved by the reporter enzyme of theantibody of the complexes, causing release of photons. Thus, as thestream of chemiluminescent substrate continuously flows through thecapillary, the resolved antibody-target complexes will continue to emitphotons. Alternatively, an excitation source may be used allowingfluorescence detection. In embodiments where chemiluminescence is used,emission is continuous for as long as the flow of chemiluminescentsubstrates is promoted, and the noise associated with stray excitationlight in fluorescence-based systems is avoided.

The photon emission events are detected by the adjacent CCD array 140and the detected events accumulated by the computer. Detection andaccumulation can be continued for a selected period of time, enablinglong detection periods to be used for sensitive detection of very smallamounts of targeted cellular molecules. When only a single labeledantibody is used, the number of events accumulated will be a measure ofthe amount of analytes in the cell or cells used to prepare the lysate.To measure the amounts of different cell proteins or molecules,different antibodies which create different antibody-target complexes atdifferent isoelectric points can be employed. By recording the number ofphoton events and the locations along the CCD array at which the eventswere detected (corresponding to the focused bands or isoelectric pointsalong the gradient field of the capillary) the photon events emanatingfrom the differently labeled analytes can be segmented. For increasedthroughput, multiple parallel capillaries or channels can be run pastone or more CCD arrays incorporated into a single instrument. In anotherembodiment, multiple antibodies labeled with different fluorescent dyeshaving spectrally resolvable signals can be used to enable multiplexedanalysis of different proteins in a single capillary.

Fluorescent standards can be read separately if desired, using the samedetector before or after the chemiluminescence signals have beencollected, by exposing the fluors to excitation light. For an allfluorescence system, the analyte and standards can be discerned by usingdifferentially excitable and detectable dyes.

While the CCD array is preferred for its ability to detect in parallelthe photon events occurring along the array, it is understood that morerestricted detection techniques may be acceptable in a given embodiment.For instance, a single photon sensor may be swept or moved along theobservation window 138 to detect the chemiluminescent or fluorescentphoton events. This approach, however, may miss an event at one point ofthe capillary when the sensor is aimed or located at a different pointof the capillary. Furthermore the use of an designed for commonlyavailable fixed window location capillary electrophoresis instruments,resolution can be deteriorated by laminar flow within the capillary, andchemiluminescent or fluorescent sensitivity would be reduced due to thelimited time that a photon source is in the observation window.

6.4 Kits

Also provided are kits for performing the methods, and for use with thesystems and devices of the present teachings. Materials used in thepresent invention include but are not limited to a fluid path,capillaries, buffer, detection agents, one or more reactive moieties,polymeric or polymerizable materials comprising one or more reactivemoieties; chemiluminescent substrates, blocking solutions, and washingsolutions. In some embodiments, the kit can further compriseimmobilization agents, ampholytes, and one or more reactive moieties. Insome embodiments, the kit can further comprise chemicals for theactivation of the reactive moiety. These other components can beprovided separately from each other, or mixed together in dry or liquidform.

7. EXAMPLES

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

7.1 Example 1

Fluorescence Detection of Green Fluorescent Protein (GFP)

Preparation of GFP sample for analysis: In a microcentrifuge tube, 40μL. of DI water, 1 μL of GFP at 1 mg/ml (Part number 632373,Beckton-Dickinson, San Jose, Calif., USA), 5 μL of bioPLUS pI 4-7(Bio-World, Dublin, Ohio), and 2 μL of ATFB-PEG cross-linking agent (2mM) were combined. The ATFB-PEG cross-linking agent consists of 15,000MW branched polyethylene glycol (product number P4AM-15, SunBio, AnyangCity, South Korea) in which each branch terminus was derivatized with anATFB (4-azido-2,3,5,6-tetrafluorobenzoic acid) functionality (productnumber A-2252, Invitrogen Corporation, Carlsbad, Calif.).

Preparation of capillary: 100μ I.D.×375μ O.D. Teflon-coated fused silicacapillary with interior vinyl coating (product number 0100CEL-01,Polymicro Technologies, Phoenix, Ariz.) was surface grafted on itsinterior with polyacrylamide containing 1 mole percent benzophenone. 4cm sections of this capillary material were cleaved from longer lengthsand used as described below.

Sample loading into capillary: The sample as prepared above was loadedinto sections of capillary prepared as described above by touching thetip of the empty capillary to the sample as illustrated in FIG. 3 b.Capillary action was sufficient to completely fill the capillary in lessthan five seconds.

Separation by isoelectric focusing (IEF): Capillaries loaded asdescribed above were placed in a capillary holder as illustrated in FIG.12 b. A 20 mM NaOH solution was placed in the cathode end and a 10 mMH₃PO₄ solution was placed in the anode end of the holder, in contactwith the electrodes and capillaries, A potential of 300 V was thenapplied for 900 seconds to facilitate isoelectric focusing, which isoften achieved within the first few minutes of this period. GFP wasresolved in a 4-7 pI gradient.

Immobilization by ultraviolet light: After the focusing period thecapillaries were irradiated for 30 seconds with UV light using an 1800Watt F300S lamp (Fusion Systems, Inc., Gaithersburg, Md.) at 5 inchesdistance from the capillaries to cause photo-crosslinking.

Washing, blocking and probing step: After immobilization as describedabove, the capillaries were removed from the capillary holder and theanodic end of each capillary was placed in contact with a TBST solutionconsisting of 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 6.8. A ≧5mmHg vacuum source was applied to the cathodic end of each capillary andTBST solution was pulled through each capillary for 5 minutes. Using thesame ≧5 mmHg vacuum source and capillary orientation, a 5% powdered skimmilk solution (w/v) in TBST was pulled through each capillary for 20minutes. Fluorescent dye labeled primary antibody solution (1:1000dilution of Alexa-555-labeled rabbit anti-GFP, Part number A-31851,Molecular Probes, Eugene, Oreg., USA in 5% milk in TBST) was thenintroduced to each capillary using the same vacuum source applied for 2minutes, followed by 10 minutes incubation with vacuum off. Thisantibody application procedure was repeated a total of 5 times. Then,the same approach was used to flush the capillary with 5% milk solutionin TBST for 20 minutes. Finally the capillaries were flushed for 5minutes with TBST, and then 2 minutes TBS (10 mM Tris-HCl, 150 mM NaCl).

Detection byfluorescence: For fluorescence detection, capillaries wereread using a Molecular Dynamics Avalanche™ scanner with excitation at532 nm and emission detection at 575 nm. The relative fluorescence unitsalong the length of the capillary as pixel number is shown in FIG. 17.The pixel number scale in FIGS. 17-22 varies because of different CCDsand positioning relative to the capillary.

7.2 Example 2

Chemiluminescence Detection of GFP

Preparation of GFP sample for analysis: In a microcentrifuge tube, 40 μLof DI water, 1 μL of GFP at 1 mg/ml (Part number 632373, BD Biosciences,San Jose, Calif., USA), 5 μL of bioPLUS pI 4-7 (Bio-World, Dublin,Ohio), and 2 μL of ATFB-PEG cross-linking agent (2 mM) were combined.The ATFB-PEG cross-linking agent consists of 15,000 MW branchedpolyethylene glycol (product number P4AM-15, SunBio, Anyang City, SouthKorea) in which each branch terminus was derivatized with an ATFB(4-azido-2,3,5,6-tetrafluorobenzoic acid) functionality (product numberA-2252, Invitrogen Corporation, Carlsbad, Calif.).

Preparation of capillary: 100μ I.D.×375μ O.D. Teflon-coated fused silicacapillary with interior vinyl coating (product number 0100CEL-01,Polymicro Technologies, Phoenix, Ariz.) was surface grafted on itsinterior with polyacrylamide containing 1 mole percent benzophenone. 4cm sections of this capillary material were cleaved from longer lengthsand used as described below.

Sample loading into capillary: The sample as prepared above was loadedinto sections of capillary prepared as described above by touching thetip of the empty capillary to the sample as illustrated in FIG. 3 b.Capillary action was sufficient to completely fill the capillary in lessthan five seconds.

Separation by isoelectric focusing (IEF): Capillaries loaded asdescribed above were placed in a capillary holder as illustrated in FIG.12 b. A 20 mM NaOH solution was placed in the cathode end and a 10 mMH₃PO₄ solution was placed in the anode end of the holder, in contactwith the electrodes and capillaries. A potential of 300 V was thenapplied for 900 seconds to facilitate isoelectric focusing, which isoften achieved within the first few minutes of this period. GFP wasresolved in a 4-7 pI gradient.

Immobilization by ultraviolet light: After the focusing period thecapillaries were irradiated for 30 seconds with UV light using an 1800Watt F300S lamp (Fusion Systems, Inc., Gaithersburg, Md.) at 5 inchesdistance from the capillaries to cause photo-crosslinking.

Washing, blocking and probing step: After immobilization as describedabove, the capillaries were removed from the capillary holder and theanodic end of each capillary was placed in contact with a TBST solutionconsisting of 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 6.8. A ≧5mmHg vacuum source was applied to the cathodic end of each capillary andTBST solution was pulled through each capillary for 5 minutes. Using thesame ≧5 mmHg vacuum source and capillary orientation, a 5% powdered skimmilk solution (w/v) in TBST was pulled through each capillary for 20minutes. Primary antibody solution (1:1000 dilution of rabbit anti-GFP,Part number A-11122, Molecular Probes, Eugene, Oreg., USA, in 5% milk inTBST) was then introduced to each capillary using the same vacuum sourceapplied for 2 minutes, followed by 10 minutes incubation with vacuumoff. This antibody application procedure was repeated a total of 5times. Then, the same approach was used to flush the capillary with 5%milk solution in TBST for 20 minutes. A secondary (2°) antibody solutionwas then applied (1:10,000 anti-rabbit HRP in 5% milk in TBST, Cat#81-6120, Zymed, South San Francisco, Calif.), again by flowing antibodysolution for 2 minutes with vacuum on followed by 10 minutes ofincubation with vacuum off, repeating a total of 5 times. Thecapillaries were then again flushed with a 5% milk solution in TBST for20 minutes. Finally the capillaries were flushed for 5 minutes withTBST, and then 2 minutes TBS (10 mM Tris-HCl, 150 mM NaCl).

Detection by chemiluminescence: For chemiluminescence detection, amixture of equal parts of SuperSignal West Femto Stable Peroxide buffer(Cat#1859023, Pierce, Rockford, Ill.) and Luminol/Enhancer solution(Cat#1859022, Pierce, Rockford, Ill.) was supplied to the capillariesand flushed through with 5mmHg vacuum. Chemiluminescence signal wascollected for 60 seconds using a CCD camera in a prototypechemiluminescence detection module produced by Cell Biosciences, U.S.Patent Application No. 60/669,694 filed Apr. 9, 2005. The relativeluminescence signal along the length of a capillary as pixel number isshown in FIG. 18.

7.3 Example 3

Fluorescent Detection of Horse Myoglobin.

Preparation of protein sample: was prepared at 20 μg/ml in ** buffer andstored at 4° C. until use, or frozen at −70° C. for long-term storage.

Preparation of sample for analysis: In a microcentrifuge tube, 40 μL ofDI water, 2 μL of 4 mg/ml purified horse myoglobin (Part number M-9267,Sigma-Aldrich, St. Louis, Mo., USA) myoglobin solution, 5 μL ofPharmalyte ampholyte pI 3-10 (Sigma, St. Louis), and 2 μL of ATFB-PEGcross-linking agent (2 mM) were combined. The ATFB-PEG cross-linkingagent consists of 15,000 MW branched polyethylene glycol (product numberP4AM-15, SunBio, Anyang City, South Korea) in which each branch terminuswas derivatized with an ATFB (4-azido-2,3,5,6-tetrafluorobenzoic acid)functionality (product number A-2252, Invitrogen Corporation, Carlsbad,Calif.).

Preparation of capillary: 100μ I.D.×375μ O.D. Teflon-coated fused silicacapillary with interior vinyl coating (product number 0100CEL-01,Polymicro Technologies, Phoenix, Ariz.) was surface grafted on itsinterior with polyacrylamide containing 1 mole percent benzophenone. 4cmsections of this capillary material were cleaved from longer lengths andused as described below.

Sample loading into capillary: The sample as prepared above was loadedinto sections of capillary prepared as described above by touching thetip of the empty capillary to the sample as illustrated in FIG. 3 b.Capillary action was sufficient to completely fill the capillary in lessthan five seconds.

Separation by isoelectricfocusing (IEF): Capillaries loaded as describedabove were placed in a capillary holder as illustrated in FIG. 12 b. A20 mM NaOH solution was placed in the cathode end and a 10 mM H₃PO₄solution was placed in the anode end of the holder, in contact with theelectrodes and capillaries. A potential of 300 V was then applied for900 seconds to facilitate isoelectric focusing, which is often achievedwithin the first few minutes of this period. Horse myoglobin wasresolved in a 3-10 pI gradient.

Immobilization by ultraviolet light: After the focusing period thecapillaries were irradiated for 30 seconds with UV light using an 1800Watt F300S lamp (Fusion Systems,

Washing, blocking and probing step: After immobilization as describedabove, the capillaries were removed from the capillary holder and theanodic end of each capillary was placed in contact with a TBST solutionconsisting of 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 6.8. A ≧5mmHg vacuum source was applied to the cathodic end of each capillary andTBST solution was pulled through each capillary for 5 minutes. Using thesame ≧5 mmHg vacuum source and capillary orientation, a 5% powdered skimmilk solution (w/v) in TBST was pulled through each capillary for 20minutes. Fluorescent labeling of goat anti-horse myoglobin primaryantibody (part number A150-103A, Bethyl Labs, Montgomery, Tex.) wasperformed by NHS ester coupling chemistry with Alexa-647 dye (partnumber A20006, Molecular Probes, Eugene, Oreg.). The primary antibodysolution (1:50 dilution of Alexa-647-labeled goat anti-horse myoglobinin 5% milk in TBST) was then introduced to each capillary using the samevacuum source applied for 2 minutes, followed by 10 minutes incubationwith vacuum off. This antibody application procedure was repeated atotal of 5 times. Then, the same approach was used to flush thecapillary with 5% milk solution in TBST for 20 minutes. Finally thecapillaries were flushed for 5 minutes with TBST, and then 2 minutes TBS(10 mM Tris-HCl, 150 mM NaCl).

Detection byfluorescence: For fluorescence detection, capillaries wereread using a Molecular Dynamics Avalanche™ scanner with excitation at633 nm and emission detection at 675 nm. The relative fluorescence unitsalong the length of a capillary as pixel number is shown in FIG. 19.

7.4 Example 4

Chemiluminescence Detection of Akt Protein from LNCaP Cell LysateSample:

Preparation of cell lysate: Cell lysate was prepared for analysis bylysing 1×10⁶ LNCaP cells (Human prostate cancer cell line) in one ml of4° C. HNTG lysis buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Triton-X100, 10% Glycerol). The resulting lysate was clarified of insolublecellular debris through centrifugation at 11,000 g for 15 min. at 4° C.The supernatant was decanted to a fresh tube and stored at 4° C. untiluse, or frozen at −70° C. for long-term storage.

Preparation of lysate sample for analysis: In a microcentrifuge tube, 40μL of DI water, 50 μL of cell lysate, 5 μL of bioPLUS pI 4-7 (Bio-World,Dublin, Ohio), and 2 μL of ATFB-PEG cross-linking agent (2 mM) werecombined. The ATFB-PEG cross-linking agent consists of 15,000 MWbranched polyethylene glycol (product number P4AM-15, SunBio, AnyangCity, South Korea) in which each branch terminus was derivatized with anATFB (4-azido-2,3,5,6-tetrafluorobenzoic acid) functionality (productnumber A-2252, Invitrogen Corporation, Carlsbad, Calif.).

Preparation of capillary: 100μ I.D.×375μ O.D. Teflon-coated fused silicacapillary with interior vinyl coating (product number 0100CEL-01,Polymicro Technologies, Phoenix, Ariz.) was surface grafted on itsinterior with polyacrylamide containing 1 mole percent benzophenone. 4cm sections of this capillary material were cleaved from. longer lengthsand used as described below.

Sample loading into capillary: The sample as prepared above was loadedinto sections of capillary prepared as described above by touching thetip of the empty capillary to the sample as illustrated in FIG. 3 b.Capillary action was sufficient to completely fill the capillary in lessthan five seconds.

Separation by isoelectric focusing (IEF): Capillaries loaded asdescribed above were placed in a capillary holder as illustrated in FIG.12 b. A 20 mM NaOH solution was placed in the cathode end and a 10 mMH₃PO₄ solution was placed in the anode end of the holder, in contactwith the electrodes and capillaries. A potential of 300 V was thenapplied for 900 seconds to facilitate isoelectric focusing, which isoften achieved within the first few minutes of this period. Akt wasresolved in a 4-7 pI gradient.

Immobilization by ultraviolet light: After the focusing period thecapillaries were irradiated for 30 seconds with UV light using an 1800Watt F300S lamp (Fusion Systems, Inc., Gaithersburg, Md.) at 5 inchesdistance from the capillaries to cause photo-crosslinking.

Washing, blocking and probing step: After immobilization as describedabove, the capillaries were removed from the capillary holder and theanodic end of each capillary was placed in contact with a TBST solutionconsisting of 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 6.8. A ≧5mmHg vacuum source was applied to the cathodic end of each capillary andTBST solution was pulled through each capillary for 5 minutes. Using thesame ≧5 mmHg vacuum source and capillary orientation, a 5% powdered skimmilk solution (w/v) in TBST was pulled through each capillary for 20minutes. Primary antibody solution (1:100 dilution of rabbit anti-AKT,sc8312, Santa Cruz Biotechnology, Santa Cruz, Calif., in 5% milk inTBST) was then introduced to each capillary using the same vacuum sourceapplied for 2 minutes, followed by 10 minutes incubation with vacuumoff. This antibody application procedure was repeated a total of 5times. Then, the same approach was used to flush the capillary with 5%milk solution in TBST for 20 minutes. A secondary (2°) antibody solutionwas then applied (1:10,000 anti-rabbit HRP in 5% milk in TBST, Cat#81-6120, Zymed, South San Francisco, Calif.), again by flowing antibodysolution for 2 minutes with vacuum on followed by 10 minutes ofincubation with vacuum off, repeating a total of 5 times. Thecapillaries were then again flushed with a 5% milk solution in TBST for20 minutes. Finally the capillaries were flushed for 5 minutes withTBST, and then 2 minutes TBS (10 mM Tris-HCl, 150 mM NaCl).

Detection by chemiluminescence: For chemiluminescence detection, amixture of equal parts of SuperSignal West Femto Stable Peroxide buffer(Cat#1859023, Pierce, Rockford, Ill.) and Luminol/Enhancer solution(Cat#1859022, Pierce, Rockford, Ill.) was supplied to the capillariesand flushed through with SmmHg vacuum. Chemiluminescence signal wascollected for 60 seconds using a CCD camera in a prototypechemiluminescence detection module produced by Cell Biosciences U.S.Patent Application No. 60/669,694 filed Apr. 09, 2005. The relativeluminescence signal along the length of a capillary as pixel number isshown in FIG. 20 and the upper panel of FIG. 22.

7.5 Example 5

Chemiluminescence Detection of Akt Protein from LNCaP Cell Lysate UsingAnti-Phospho-S473-Antibody:

Preparation of cell lysate: Cell lysate was prepared for analysis bylysing 1×10⁶ LNCaP cells (Human prostate cancer cell line) in one ml of4° C. HNTG lysis buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Triton-X100, 10% Glycerol). The resulting lysate was clarified of insolublecellular debris through centrifugation at 11,000 g for 15 min. at 4° C.The supernatant was decanted to a fresh tube and stored at 4° C. untiluse, or frozen at −70° C. for long-term storage.

Preparation of lysate sample for analysis: In a microcentrifuge tube, 40μL of DI water, 50 μL of cell lysate, 5 μL of Pharmalyte ampholyte pI3-10 (Sigma, St. Louis), and 2 μL of ATFB-PEG cross-linking agent (2 mM)were combined. The ATFB-PEG cross-linking agent consists of 15,000 MWbranched polyethylene glycol (product number P4AM-15, SunBio, AnyangCity, South Korea) in which each branch terminus was derivatized with anATFB (4-azido-2,3,5,6-tetrafluorobenzoic acid) functionality (productnumber A-2252, Invitrogen Corporation, Carlsbad, Calif.).

Preparation of capillary: 100μ I.D.×375μ O.D. Teflon-coated fused silicacapillary with interior vinyl coating (product number 0100CEL-01,Polymicro Technologies, Phoenix, Ariz.) was surface grafted on itsinterior with polyacrylamide containing 1 mole percent benzophenone. 4cmsections of this capillary material were cleaved from longer lengths andused as described below.

Sample loading into capillary: The sample as prepared above was loadedinto sections of capillary prepared as described above by touching thetip of the empty capillary to the sample as illustrated in FIG. 3 b.Capillary action was sufficient to completely fill the capillary in lessthan five seconds.

Separation by isoelectric focusing (IEF): Capillaries loaded asdescribed above were placed in a capillary holder as illustrated in FIG.12 b. A 20 mM NaOH solution was placed in the cathode end and a 10 mMH₃PO₄ solution was placed in the anode end of the holder, in contactwith the electrodes and capillaries. A potential of 300 V was thenapplied for 900 seconds to facilitate isoelectric focusing, which isoften achieved within the first few minutes of this period. Protein wasresolved in a 4-7 pI gradient

Immobilization by ultraviolet light: After the focusing period thecapillaries were irradiated for 30 seconds with UV light using an 1800Watt F300S lamp (Fusion Systems, Inc., Gaithersburg, Md.) at 5 inchesdistance from the capillaries to cause photo-crosslinking.

Washing, blocking and probing step: After immobilization as describedabove, the capillaries were removed from the capillary holder and theanodic end of each capillary was placed in contact with a TBST solutionconsisting of 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 6.8. A ≧5mmHg vacuum source was applied to the cathodic end of each capillary andTBST solution was pulled through each capillary for 5 minutes. Using thesame ≧5 mmHg vacuum source and capillary orientation, a 5% powdered skimmilk solution (w/v) in TBST was pulled through each capillary for 20minutes. Primary antibody solution (1:100 dilution of rabbitanti-phospho-S473 AKT, Part number 4051, Cell Signaling Technologies,Beverly, Mass., USA, in 5% milk in TBST) was then introduced to eachcapillary using the same vacuum source applied for 2 minutes, followedby 10 minutes incubation with vacuum off. This antibody applicationprocedure was repeated a total of 5 times. Then, the same approach wasused to flush the capillary with 5% milk solution in TBST for 20minutes. A secondary (2°) antibody solution was then applied (1:10,000anti-rabbit HRP in 5% milk in TBST, Cat# 81-6120, Zymed, South SanFrancisco, Calif.), again by flowing antibody solution for 2 minuteswith vacuum on followed by 10 minutes of incubation with vacuum off,repeating a total of 5 times. The capillaries were then again flushedwith a 5% milk solution in TBST for 20 minutes. Finally the capillarieswere flushed for 5 minutes with TBST, and then 2 minutes TBS (10 mMTris-HCl, 150 mM NaCl).

Detection by chemiluminescence: For chemiluminescence detection, amixture of equal parts of SuperSignal West Femto Stable Peroxide buffer(Cat#1859023, Pierce, Rockford, Ill.) and Luminol/Enhancer solution(Cat#1859022, Pierce, Rockford, Ill.) was supplied to the capillariesand flushed through with 5 mmHg vacuum. Chemiluminescence signal wascollected for 60 seconds using a CCD camera in a prototypechemiluminescence detection module produced by Cell Biosciences, U.S.Patent Application No. 60/669,694 filed Apr. 09, 2005. The relativeluminescence signal along the length of a capillary as pixel number isshown in FIG. 21 and the lower panel of FIG. 22.

FIG. 22 compares the chemiluminescence signals generated in FIGS. 21 and22 illustrating the resolving of phosphorylated and non-phosphorylatedforms of the Akt protein by a single, total-protein-specific antibody inthe upper panel FIG. 21. The lower panel FIG. 21, showing the signalgenerated using the phospho-specific antibody, indicates that the peaksresolved in box A are phosphorylated forms, while those resolved in boxB are non- phosphorylated at serine 473.

The foregoing descriptions of specific embodiments of the presentinvention have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and obviously manymodifications and variations are possible in light of the aboveteaching. The embodiments were chosen and described in order to bestexplain the principles of the invention and its practical application,to thereby enable others skilled in the art to best utilize theinvention and various embodiments with various modifications as aresuited to the particular use contemplated. It is intended that the scopeof the invention be defined by the Claims appended hereto and theirequivalents.

All patents, patent applications, publications, and references citedherein are expressly incorporated by reference to the same extent as ifeach individual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. A method of detecting at least one analyte in a sample, characterizedin that: one or more analytes are resolved in a fluid path and saidanalytes are immobilized in said fluid path and detection agents areconveyed through the fluid path which bind to or interact with theanalytes and permit detection of said immobilized analytes.
 2. Themethod of claim 1, wherein the fluid path comprises a capillary.
 3. Themethod of claim 1, further comprising a plurality of fluid paths,wherein said fluid paths are capillaries.
 4. The method of claim 1,wherein the fluid path comprises a channel in a microfluidic device. 5.The method of claim 1, further comprising a plurality of fluid paths,wherein said fluid paths are channels in a microfluidic device.
 6. Themethod of claim 1, wherein said analytes are resolved byelectrophoresis.
 7. The method of claim 1, wherein said analytes areresolved by isoelectric focusing, gel electrophoresis, or electrokineticchromatography.
 8. The method of claim 1, wherein said analytes areimmobilized in said fluid path by isoelectric focusing.
 9. The method ofclaim 1, wherein said fluid path further comprises one or more reactivemoieties capable of immobilizing said analytes in said fluid path,wherein immobilizing comprises activating said reactive moiety ormoieties.
 10. The method of claim 9, wherein said activating comprisesphotoactivation, chemical activation or thermal activation of saidreactive moiety or moieties.
 11. The method of claim 1, wherein thedetection agents are conveyed through the fluid path by hydrodymic flow,electroendosmotic flow, or electrophoresis.
 12. The method of claim 1,wherein said detection agents comprise antibodies, peptides, proteins,enzyme substrates, transition state analogs, cofactors, nucleotides,polynucleotides, aptamers, lectins, small molecules, ligands,inhibitors, drugs, or labeled molecules thereof, or chemiluminescentsubstrates.
 13. The method of claim 1, wherein said detection agentscomprise chemiluminescent labeled antibodies.
 14. The method of claim 1,wherein said detection agents comprise fluorescent labeled antibodies.15. The method of claim 1, wherein the detection of said analytescomprises detecting a chemiluminescent signal.
 16. The method of claim1, wherein detection of said analytes comprises detecting a fluorescentsignal.
 17. The method of claim 1, wherein detection of said analytes isby a photodetector.
 18. The method of claim 1, wherein detection of saidanalytes is by an array of photodetectors.
 19. The method of claim 1,wherein detection of said analytes is by a charged coupled device (CCD)array.
 20. A method of detecting at least one protein of interest in asample, comprising: a) resolving one or more proteins in a capillary; b)photoimmobilizing said proteins in said capillary; and c) contactingsaid immobilized proteins with antibodies to form one or moreantibody-protein complexes in the capillary; and d) detecting saidprotein.
 21. The method of claim 20, wherein said antibodies comprise alabel.
 22. The method of claim 20, further comprising contacting saidantibody-protein complex with a labeled antibody.
 23. The method ofclaim 20, wherein said detecting comprises detecting a chemiluminescentsignal.
 24. The method of claim 20, wherein said detecting comprisesdetecting a fluorescent signal.
 25. A method of detecting at least oneprotein in a sample, comprising: a) resolving one or more proteins in acapillary; wherein said capillary comprises one or more photoreactivegroups and optionally polymeric or polymerizable materials comprisingone or more photoreactive groups; b) photoimmobilizing said protein insaid capillary; and c) contacting antibodies to said photoimmobilizedprotein to form one or more antibody-target complexes in the capillary;and d) detecting said protein.
 26. A method of detecting at least oneprotein in a sample, comprising: a) concentrating one or more proteinsin a fluid path; b) immobilizing said proteins in said fluid path; andc) contacting said immobilized protein with antibodies to form one ormore antibody-protein complexes in said fluid path; and d) detectingsaid protein.
 27. A system for detecting at least one analyte ofinterest in a sample, comprising: a) a fluid path comprising one or morereactive groups capable of immobilizing said analytes; b) a power supplyfor applying a voltage along the fluid path capable of resolving saidanalytes in said fluid path; and c) a detector capable of detecting saidanalytes in said fluid path.
 28. The system of claim 27, wherein saiddetector comprises a photodetector.
 29. The system of claim 27, whereinsaid detector comprises an array of photodetectors.
 30. The system ofclaim 27, wherein said detector is by a charged coupled device (CCD)array.
 31. The system of claim 27, further comprising a cell lysingdevice.
 32. A kit for detecting at least one analyte in a samplecomprising a fluid path comprising one or more reactive moieties,buffer, and detection agents.